Biochemistry I Fall Term, 2000

Lecture 33: Oxidative Phosphorylation

Assigned reading in Campbell: Chapter 16
Redox Chemistry in Biological Processes: Review of Chapter 11.8-11.11 topics.

Take the Review Quiz on Lecture 33 concepts.

Synthesis of ATP from the Proton Gradient: an animation of the F_oF₁ ATP synthesis cycle.
F₁ ATPase: Chime page showing the "motor" and its coiled-coil shaft (450 KB).
ATP Synthase: A detailed lecture by Antony Crofts, University of Illinois.
(Be sure to watch the movie of the rotating F₁ ATPase.)

Four Key Complexes in Electron Transport
The following complexes are localized to the inner mitochondrial membrane.

• An integral membrane protein complex.
• Contains flavin mononucleotide (FMN) covalently attached to proteins; also contains iron-sulfur proteins.
• Two electrons from NADH are transferred to Coenzyme Q.
  1. CoQ is mobile within the inner membrane.
  2. Cycles between CoQ <=> CoQH₂.
• Two protons are pumped from the inside (matrix) to the intermembrane space.

• An integral membrane protein complex.
• Succinate dehydrogenase of the citric acid cycle is part of this complex.
• Two electrons from FADH₂ are transferred to CoQ.
• Electrons are passed to cyctochromes.
• Does not pump any protons.

• An integral membrane protein complex.
• Transfers electrons from CoQ to cytochrome c one electron at a time.
• Four protons are pumped for each pair of electrons transferred.

• An integral membrane protein complex.
• Contains cyt a and cyt a₃
• Accepts one electron at a time from cytochrome c.
• Donates a total of four electrons/O₂.
• Site of oxygen reduction to water.
  1. Produces 2 water molecules/O₂ molecule.
  2. Pumps an additional two protons across the membrane.

ATP Synthesis

ATP synthesis occurs in the mitochondrial matrix.
The endergonic formation of ATP from ADP + P_i is driven by two factors related to the electron transport described above.

1. The proton gradient.
   The transport of electrons caused protons to be pumped out of the matrix space. The resulting difference in pH can be coupled to ATP synthesis.
2. The membrane potential.
   The transport of H⁺ out leaves the matrix at a negative electrical potential relative to the cytosol. The resulting difference in membrane potential can be coupled to ATP synthesis.

DG = 2.3RTDpH + ZFDY, where:

DpH = pH_out - pH_in;

Z = the proton charge;

F = the Faraday constant (96,494 J/volt-mol);

DY = the membrane potential (volts).

For typical values of these terms (DpH = 1.0, and DY = -150 mV at 37°C)
DG = -5.9 kJ/mol - 14.5 kJ/mol
DG = -20.4 kJ/mol

The "ATP synthase motor" (F_oF₁ATPase) converts the free energy of the proton gradient to chemical energy in the form of ATP.

The F_o Complex

• Membrane-spanning, multiprotein complex.
• Responsible for coupling the movement of each proton to 120° rotations of the F₁ portion.

The F₁ Complex

• Five different subunits: a₃b₃gde
• Attached to F_o, it protrudes into the mitochondrial matrix.
• The b subunits are asymmetric due to their interactions with the F_o
.
1. One b subunit has very low affinity for both ADP and ATP.
2. One b subunit has high affinity for ADP and P_i.
3. One b subunit has high affinity for ATP.
ADP or ATP can only be released from the low affinity subunit
• The g subunit is the shaft at the center of the a₃b₃ disk.

• Every time a proton is pumped, the F₁ subunit rotates 120°.
• The actual synthesis (formation of the bond between ADP and P_i is catalyzed by conformational changes of the enzyme that occur as a consequence of the rotation.
• The key point is that the rotation moves the b subunit that contains ADP + P_ito a new position. In this new position the b subunit would rather bind ATP, and thus catalyzes the formation of a ATP from the bound ADP and P_i. The newly-formed ATP is released with the transport of an additional proton.
• Three protons must be transported to make one ATP.

The net gains by oxidative phosphorylation are:

Coupling Electron Transport to ATP Synthesis

• Mitchell's chemiosmotic theory.
• Requirement for an intact (i.e. ion-impermeable) membrane-enclosed space.
• Requirement for a proton concentration gradient (DpH).
• The membrane potential is a driving force.
• ATP synthase is physically separate from electron flow (and proton pumping out of the mitochondrion).
  1. Intact mitochondria.
  2. Inside-out vesicles.
  3. Reconstituted vesicles with bacteriorhodopsin as H⁺ pumps.
• Uncouplers permit protons to flow in without ATP synthesis.
  1. Dinitrophenol (DNP) shuttles H⁺ across the membrane.
  2. Antibiotics create ion channels, e.g. gramicidin A.
  3. Beneficial uncouplers result in heat production where it is needed.

Respiratory Inhibitors Act at Selected Sites.

• Complex I: Rotenone, a fish poison, is harmless to humans.
• Complex II: Antimycin A.
• Complex IV: CN^-, N₃^-, and CO.

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